2. Biological Control: Develop new and improve existing biological control
methods for ACP and sharpshooter species.
2a. Establish natural enemies of ACP in Florida.
2b. Identify new viral pathogens of ACP and sharpshooter species, and
evaluate the potential of these as management strategies.

1b.Approach (from AD-416):
Behavioral assays and antennogram (EAG/EAD) methods will be developed for key citrus pests to identify conspecific odors that mediate mate location, recognition or aggregation. Methods for monitoring and control using these compounds will be developed. Host and nonhost compounds will be identified that affect host selection, location or repellency through the use of gas chromatography (GC), GC-EAD, and GC-mass spectroscopy (GC-MS). To increase natural biological control of the Asian citrus psyllid, haplotypes of the parasitoid Tamarixia radiata will be characterized and released as deemed appropriate. Viral pathogens of Asian citrus psyllid and sharpshooter species will be identified by genomic methods and evaluated as management strategies. Existing citrus germplasm will be screened for host plant resistance to Asian citrus psyllids under greenhouse and field conditions. Genes and proteins involved in key biological processes such as salivary sheath formation will be studied to develop novel control strategies that block these processes.

A setback to a delivery system for citrus leafminer (CLM) pheromone occurred when formulation modification resulted in loss of activity. Field trials identified this failure and re-established trap catch disruption using rubber dispensers. Dispensers were >4 times as effective as the best performing formulation from previous years. The manufacturer now provides rubber dispensers for validation trials. Transects showed leafminer movement up to 1.2 km from infested citrus.

A patent for a Diaprepes root weevil pheromone was received; exclusive rights were licensed to Citrus Research and Development Foundation. A second putative pheromone is being evaluated in combination with plant volatiles. No attractant has yet been demonstrated.

Related to the second objective, releases and impact assessments of parasitoids for Asian citrus psyllid (ACP) control in urban areas continued. An experiment initiated in organic citrus evaluated repetitive parasitoid releases.
Related to the third objective, most accessions of Poncirus trifoliata and hybrids were resistant to Asian citrus psyllid (ACP) colonization. Volatile components of resistant and susceptible genotypes are being studied. Adults confined to some P. trifoliata accessions died sooner than adults on susceptible genotypes; P. trifoliata secondary metabolites may interfere with ACP biology.

Progress was made towards Objective 3b for Asian citrus psyllid (ACP) and glassy-winged sharpshooter (GWSS). Improved ACP genome assembly incorporated short- and long-read sequencing resulting in a more accurate genome. Microbial sequences were removed from the short-read sequence to produce a metagenome dataset. This resulted in a draft genome of the endosymbiotic bacterium Wolbachia-Diaphorina. The GWSS effort completed sequencing at Baylor Human Genome Sequencing Center, Houston, Texas in collaboration with the i5K effort to sequence insect genomes. This is the first genome from any leafhopper.

4.Accomplishments
1.
Discovery of pheromones from the Diaprepes root weevil. Agricultural Research Service entomologists at Fort Pierce, Florida in collaboration with ARS colleagues at Beltsville, Maryland and Gainesville, Florida received a provisional patent for a male-produced pheromone for the Diaprepes root weevil that is expected to be licensed to the Citrus Research and Development Foundation. Additional compounds have been identified that elicit antennal responses and behavioral orientation that may play a role in orientation by both sexes. Deciphering the behavioral repertoire of Diaprepes in response to blends of these odors should result in new lures, traps and control methods for this important pest of citrus.

2.
A new product for citrus leafminer pheromone release. Agricultural Research Service entomologists at Fort Pierce, Florida in collaboration with colleagues at the University of Florida, Lake Alfred and private industry (ISCA Technologies, Inc.) demonstrated prolonged and high levels of disruption of orientation by the citrus leafminer to its sex pheromone using a solid rubber release device. Trap catch disruption of over 30 weeks was achieved at 3 locations in Florida.

3.
Discovery of citrus germplasm resistant to Asian citrus psyllid. Researchers at the USDA, ARS, USHRL in Fort Pierce, FL showed that most accessions of the genus species Poncirus trifoliata as well as a number of its hybrids have natural resistance to the Asian citrus psyllid. Two types of resistance were identified. Resistant types (antixenosis) greatly reduce infestation levels of the psyllid, a trait that may be related to volatiles used by the psyllid to find and infest plants or the presence of a volatile that repels the psyllid. The other resistant type (antibiosis) results in reduced longevity of adult psyllids, possibly related to the presence of secondary plant metabolites. Poncirus trifoliata can be crossed with Citrus species using traditional breeding methods, thus it may be possible to identify traits conferring resistance and to breed these traits into Citrus spp.

4.
Single gene proteins inhibit Asian citrus pysllid salivary sheath formation and degrade preformed sheaths. Researchers at the USDA, ARS, USHRL in Fort Pierce, FL discovered specific proteins that block salivary sheath formation, a potentially novel method of control of Asian citrus psyllid (ACP) and citrus greening disease using transgenic citrus strategies. Discovery of double-stranded ribonucleic acids (dsRNAs) that induce ACP mortality when fed in artificial diets and the subsequent discover of how citrus engineered to produce ACP gene targeting dsRNA can block Citrus Greening transmission. Researchers at the USDA, ARS, U.S. Horticultural Research Laboratory have shown that an RNAi-based strategy of ACP mRNA knock-down has the potential to block ACP feeding on citrus and inhibit the transmission of the bacterium that causes Citrus Greening disease. This work is being advanced to determine its commercial application.

5.
First leafhopper genome completed. Agricultural Research Service entomologists at Fort Pierce, Florida established an international consortium to annotate the genome of the glassy-winged sharpshooter, Homalodisca vitripennis, selected as an i5K project collaboration with the researchers at the Baylor College of Medicine, Human Genome Center, Houston, TX. Genomic DNA from male, female, nymphs, and eggs, was sequenced. The results advance the understanding of leafhopper biology and pathogen transmission, setting the foundation for development of new leafhopper control measures such as ribonucleic acid (RNA)interference.

6.
Asian citrus psyllid genome now available online. Agricultural Research Service entomologists at Fort Pierce, Florida led an international psyllid genome consortium that completed an improved genome assembly of the Asian citrus psyllid in 2013. The improved datasets created a unigene set of 18,300 predicted genes. The genome and transcriptome datasets provide a foundation for further investigation of functional psyllid genomics and serves as a reference genome for sequencing other psyllid species. The genome provides critical information on pathogen transmission and insecticide resistance to develop methods for psyllid control and plant improvement aimed at reducing losses to citrus greening disease.

7.
Metagenome of Asian citrus psyllid elucidates Wolbachia-Diaphorina endosymbiont. Agricultural Research Service entomologist at Fort Pierce, Florida co-led an international psyllid metagenome consortium effort which sequenced the endosymbiotic bacterium Wolbachia-Diaphorina, Florida strain. Multiple gene analysis demonstrated that the psyllid in Florida was most closely related to psyllid populations collected from northern India. This identifies the point of origin and locations to be explored for biological control agents.

8.
Discovery of double-stranded ribonucleic acid (dsRNA) molecules to control Asian citrus psyllids. Researchers at the USDA, ARS, U.S. Horticultural Research Laboratory in Fort Pierce, Florida in collaboration with scientist from EMBRAPA, Brazil, showed that psyllid mortality was significantly increased when they fed on citrus plants infused with dsRNA molecules that have a sequence identical to either of two psyllid genes. This non-transgenic approach of RNAi for pest and disease management offers the possibility of targeting psyllids in a highly specific manner that safeguards nontarget beneficial insects, honey bees and other pollinating species.